Endothelial Cells Mediated by UCP2 Control the Neurogenic‐to‐Astrogenic Neural Stem Cells Fate Switch During Brain Development

Abstract During mammalian cortical development, neural stem/progenitor cells (NSCs) gradually alter their characteristics, and the timing of generation of neurons and glial cells is strictly regulated by internal and external factors. However, whether the blood vessels located near NSCs affect the neurogenic‐to‐gliogenic transition remain unknown. Here, it is demonstrated that endothelial uncoupling protein 2 (UCP2) deletion reduces blood vessel diameter and affects the transition timing of neurogenesis and gliogenesis. Deletion of endothelial UCP2 results in a persistent increase in astrocyte production at the postnatal stage. Mechanistically, the endothelial UCP2/ROS/ERK1/2 pathway increases chymase‐1 expression to enhance angiotensin II (AngII) secretion outside the brain endothelium. The endotheliocyte‐driven AngII‐gp130‐JAK‐STAT pathway also regulates gliogenesis initiation. Moreover, endothelial UCP2 knockdown decreases human neural precursor cell (hNPC) differentiation into neurons and accelerates hNPC differentiation into astrocytes. Altogether, this work provides mechanistic insights into how endothelial UCP2 regulates the neurogenic‐to‐gliogenic fate switch in the developing neocortex.

. Endothelial UCP2 deletion reduces the blood vessel diameter.
(A) Confocal immunofluorescence image of IB4 and GFAP at E18. The right is higher magnification images and 3D reconstructions of z-stacks of brain vessels and astrocytes.
(C) Confocal immunofluorescence image of IB4 and UCP2 in the UCP2 WT and UCP2 ECKO isolated brain endothelial cells. Scale bar, 50μm.
(D) Quantification of UCP2 relative expression and showing the expression of UCP2 was depleted in UCP2 ECKO brain endothelial cells.***P<0.001(mean ± SEM, unpaired two-tailed Student's t test, n =5 each group from 3 independent experiments).
(E)Western blot analysis of UCP2 expression levels in the UCP2 WT and UCP2 ECKO isolated brain endothelial cells. β-actin was detected as loading control.
(F) Confocal immunofluorescence image of IB4 and PDGFRβ in the UCP2 WT and UCP2 ECKO cortical sections at P2. The right magnification images showing a decreased mean diameter in UCP2 ECKO cortical sections. Scale bars, 100μm (left),10μm (right).
(J) Quantification of the PDGFRβ + pericytes coverage showing no changes between UCP2 WT and UCP2 ECKO mice. n.s., not significant (mean ± SEM, unpaired two-tailed Student's t test, n =4 mice each group).
(A) Confocal immunofluorescence image of IB4 and Claudin-5 in the UCP2 WT and UCP2 ECKO mice. Scale bar, 20μm.
(C) Confocal immunofluorescence image of IB4 and ZO-1 in the UCP2 WT and UCP2 ECKO mice. Scale bar, 20μm.
(E) Confocal images of IB4 and Cad-A555 showing no cadaverine extravasation in UCP2 WT and UCP2 ECKO brain cortex from the blood vessels. Scale bar, 100μm.
(F) Confocal images of Col IV showing no statistical difference between UCP2 WT and UCP2 ECKO brain cortex. Scale bar, 100μm.
(G) Quantification of the fluorescence intensity of Col IV staining showing no statistical difference between UCP2 WT and UCP2 ECKO mice. n.s., not significant. (mean ± SEM, unpaired two-tailed Student's t test, n =4 mice each group).
Data are represented as means ± SEM. unpaired two-tailed Student's t test; At least three biological replicates are shown. n.s., not significant. ***P<0.001. Figure S3. Endothelial UCP2 deletion promotes astrocyte progenitor production.   (K) Western blot analysis of the expression levels of synaptic marker p-CREB and synaptophysin at P2. β-actin was detected as loading control.
(L) Statistics of relative intensity of p-CREB and synaptophysin. n.s., not significant.
(M) Western blot analysis of the expression levels of synaptic marker p-CREB and synaptophysin at P8. β-actin was detected as loading control.
(N) Statistics of relative intensity of p-CREB and synaptophysin. n.s., not significant.
Data are represented as means ± SEM. At least three biological replicates are shown. unpaired two-tailed Student's t test; n.s., not significant. *P<0.05, **P<0.01. Data are represented as means ± SEM. unpaired two-tailed Student's t test; n.s., not significant. n.s., not significant.  (I) Confocal immunofluorescence images of GFAP and MAP2 of neural precursor cells after H2O or Ang II treatments. Scale bars, 50μm.

Figure S8. Ang II accelerates human NPCs differentiation toward astrocytes.
(A) Confocal immunofluorescence images of MAP2 and GFAP of human NPCs after H2O or Ang II treatments. Scale bar, 50μm.
(B) Quantification of the percent of MAP2 + neurons showing decreased MAP2 + neurons s in human NPCs after H2O or Ang II treatments. *P<0.05 (mean ± SEM, unpaired two-tailed Student's t test, control n =7, Ang II n=4 from 3 independent experiments).
(C) Quantification of the percent of GFAP + astrocytes showing increased GFAP + astrocytes in human NPCs after H2O or Ang II treatments. *P<0.05 (mean ± SEM, unpaired two-tailed Student's t test, n =7 each group from 3 independent experiments).
(D) Model showing how endothelial UCP2 regulates the neurogenic-to-astrogenic fate switch through Ang II in the developing cortex.
Data are represented as means ± SEM. unpaired two-tailed Student's t test; At least three biological replicates are shown. *P<0.05.